Bacterium having ability to produce L-glutamic acid, L-proline or L-arginine and method for producing L-glutamic acid, L-proline or L-arginine

ABSTRACT

L-Glutamic acid, L-proline or L-arginine is produced by culturing a bacterium belonging to the genus  Escherichia , which is L-isoleucine auxotrophic and has ability to produce L-glutamic acid, L-proline or L-arginine, in a medium containing L-isoleucine, to produce and accumulate L-glutamic acid, L-proline or L-arginine in a culture, and collecting L-glutamic acid, L-proline or L-arginine from the culture.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a divisional of U.S. application Ser. No.09/953,298, filed on Sep. 17, 2001, which claims priority to RU2000124295, filed on Sep. 26, 2000.

BACKGROUND OF THE INVENTION

The present invention relates to techniques in the field of microbialindustry. In particular, the present invention relates to a method forproducing L-glutamic acid, L-proline or L-arginine by fermentation, anda. bacterium used in the method. L-Glutamic acid, L-arginine andL-proline are important as food, medicine and the like.

L-Arginine and L-proline are synthesized by E. coli cells from a commonprecursor, L-glutamic acid. Therefore, the level of the production ofL-arginine or L-proline depends on availability of their commonprecursor, L-glutamic acid.

There are known strains of E. coli having an increased level ofL-glutamic acid synthesis. In particular, mutants which are derived fromE. coli K12 strain and are deficient or decrease in 2-ketoglutaratedehydrogenase activity, can produce L-glutamic acid with a fairly highproductivity (U.S. Pat. Nos. 5,393,671 and 5,908,768).

It is known as well that some E. coli mutants can produce L-arginine andL-proline. They were obtained as mutants resistant to analogs of thoseamino acids and by cloning of some genes important for theirbiosynthesis (UK patent publication No. 2080825A).

SUMMARY OF THE INVENTION

An object of the present invention is to provide a novel bacteriumhaving ability to produce L-glutamic acid, L-proline or L-arginine and amethod for producing L-glutamic acid, L-proline or L-arginine by usingthe bacterium having ability to produce L-glutamic acid, L-proline orL-arginine.

The present inventors found that L-isoleucine auxotrophs of E. coli,having a deficiency in an ilvA gene, produced L-glutamic acid. Besides,strains having a deficiency in an ilvA gene, can be used as parentstrains for breeding of producers of L-proline and L-arginine. In otherwords, the present inventors found that L-isoleucine auxotrophy can beused for improvement of producers of L-glutamic acid, L-proline orL-arginine. Thus, the present invention has been accomplished.

The present invention provides the followings.

-   (1) An Escherichia bacterium which is L-isoleucine auxotrophic and    has ability to produce L-glutamic acid, L-proline or L-arginine.-   (2) The Escherichia bacterium according to (1), which is deficient    in any one of activities of L-isoleucine biosynthesis enzymes.-   (3) The Escherichia bacterium according to (2), which is deficient    in threonine deaminase activity.-   (4) The Escherichia bacterium according to any one of (1) to (3),    which is Escherichia coli.-   (5) A method for producing L-glutamic acid, L-proline or L-arginine,    which comprises culturing the Escherichia bacterium as defined in    any one of (1) to (4) in a medium containing L-isoleucine, to    produce and accumulate L-glutamic acid, L-proline or L-arginine in a    culture and collecting L-glutamic acid, L-proline or L-arginine from    the culture.

DETAILED DESCRIPTION OF THE INVENTION

<1> Bacterium of the Present Invention

The bacterium of the present invention is a bacterium belonging to thegenus Escherichia, which is L-isoleucine auxotrophic and has ability toproduce L-glutamic acid, L-proline or L-arginine. An example of theEscherichia bacterium is Escherichia coli.

The expression “a bacterium has ability to produce L-glutamic acid,L-proline or L-arginine” means that the bacterium accumulates asignificant amount of L-glutamic acid, L-proline or L-arginine in amedium when the bacterium is cultured in the medium, or increases thecontent of L-glutamic acid, L-proline or L-arginine in the bacterium.The expression “a bacterium is L-isoleucine auxotrophic” means that thebacterium requires L-isoleucine (usually, not less than 10 mg/l) in amedium for growth.

The bacterium of the present invention produces at least L-glutamicacid, L-proline or L-arginine and may produce two or more types ofL-amino acids.

The bacterium of the present ivention can be obtained by impartingL-isoleucine auxotrophy to an Escherichia bacterium having ability toproduce L-glutamic acid, L-proline or L-arginine, or by impartingability to produce L-glutamic acid, L-proline or L-arginine to anL-isoleucine auxotrophic Escherichia bacterium.

In order to impart the L-isoleucine auxotrophy, there can be used amethod comprising subjecting Escherichia bacteria to mutagenesis,allowing the Escherichia bacteria to form colonies on an agar mediumcontaining L-isoleucine, replicating the colonies to an agar medium notcontaining L-isoleucine, and selecting strains that cannot grow on theagar medium not containing L-isoleucine. The mutagenesis includes UVirradiation and treatments with mutagenesis agents used for usualmutagenesis treatments such as N-methyl-N′-nitro-N-nitrosoguanidine(NTG) and nitrous acid. Alternatively, naturally occurring mutants maybe selected.

The isoleucine auxotrophy is preferably due to a deficiency in any ofL-isoleucine biosynthetic enzyme activities (activities of enzymescatalyzing reactions of L-isoleucine biosynthesis). The L-isoleucinebiosynthetic enzymes includes threonine deaminase, acetohydroxyacidsynthase, acetohydroxy-acid isomeroreductase, dihydroxy-aciddehydratase. It is preferred that threonine deaminase activity isdeficient. The expression “activity is deficient” usually means that theintracellular activity of the enzyme is lower than that of a wild typestrain, and when a strain in which the activity of the enzyme isdeficient is obtained by modification using gene recombinant techniquesor the like, the intracellular activity of the enzyme is lower than thatof the strain before the modification.

In order to obtain the deficiency of the enzyme activity as mentionedabove, a mutation causing the deficiency of the enzyme activity can beintroduced into a gene encoding the enzyme by a conventional mutagenesistechnique or genetic engineering technique.

Examples of the mutagenesis technique include, for example, the methodutilizing irradiation of X-ray or ultraviolet light, the methodutilizing treatment with a mutagenic agent such asN-methyl-N′-nitro-N-nitrosoguanidine and the like. The site of gene towhich a mutation is introduced may be a coding region encoding an enzymeprotein, or an expression regulatory region such as a promoter.

Examples of the genetic engineering technique include, for example,genetic recombination, genetic transduction, cell fusion and the like.For example, a drug resistance gene is inserted into a target gene toproduce a functionally inactivated gene (defective gene). Then, thisdefective gene is introduced into a cell of a microorganism belonging tothe genus Escherichia, and the target gene on a chromosome is replacedwith the defective gene by homologous recombination (gene disruption).

Whether a microorganism decreases in an activity of a target enzyme oris deficient in the activity, and degree of the decrease of the activitycan be determined by measuring the enzyme activity of a bacterial cellextract or a purified fraction of a candidate strain, and comparing itwith that of a wild type strain or a parent strain. Depending on thetarget enzyme, a target variant can be selected based on a phenotype ofthe variant.

In order to impart ability to produce L-glutamic acid, L-proline orL-arginine, there can be used methods conventionally adopted forbreeding Escherichia bacteria or the like, such as those methods forobtaining auxotrophic mutant strains, strains resistant to L-amino acidanalogues or metabolic control mutant strains, and methods for producingrecombinant strains wherein L-amino acid biosynthetic enzyme activitiesare enhanced (see “Amino Acid Fermentation”, the Japan ScientificSocieties Press [Gakkai Shuppan Center], 1st Edition, published on May30, 1986, pp. 77 to 100). In breeding of amino acid-producing bacteria,the characteristic such as auxotrophy, L-amino acid analogue resistanceand metabolic control mutation may be imparted alone or in combinationof two or more. The L-amino acid biosynthetic enzyme activity may beenhanced alone or in combination of two or more. Further, imparting ofthe characteristic such as auxotrophy, L-amino acid analogue resistanceand metabolic control mutation may be combined with enhancement of theL-amino acid biosynthesis enzyme activity.

For example, L-glutamic acid-producing bacteria can be bred as mutantsexhibiting auxotrophy for oleic acid or the like.

Also, L-glutamic acid-producing ability can be imparted by, for example,introducing a DNA that codes for any one of enzymes including glutamatedehydrogenase (Japanese Patent Application Laid-open (Kokai)61-268185/1986), glutamine synthetase, glutamate synthase, isocitratedehydrogenase (Japanese Patent Application Laid-open (Kokai) Nos.62-166890/1987 and 63-214189/1988), aconitate hydratase (Japanese PatentApplication Laid-open (Kokai) No. 62-294086/1987), citrate synthase(Japanese Patent Application Laid-open (Kokai) Nos. 62-201585/1987 and63-119688/1988), phosphoenolpyruvate carboxylase (Japanese PatentApplication Laid-open (Kokai) Nos. 60-87788/1985 and 62-55089/1987),pyruvate dehydrogenase, pyruvate kinase, phosphoenolpyruvate synthase,enolase, phosphoglyceromutase, phosphoglycerate kinase,glyceraldehyde-3-phosphate dehydrogenase, triose phosphate isomerase,fructose bisphosphate aldolase, phosphofructokinase (Japanese PatentApplication Laid-open (Kokai) No. 63-102692/1988), glucose phosphateisomerase, glutamine-oxoglutarate aminotransferase (WO99/07853) and soforth.

Further, the bacterium of the present invention may be made to bedeficient in activity of an enzyme that catalyzes a reaction forgenerating a compound other than L-glutamic acid by branching off fromthe biosynthetic pathway of L-glutamic acid. The enzyme that catalyzesthe reaction for generating the compound other than L-glutamic acid bybranching off from the biosynthetic pathway L-glutamic acid includeα-ketoglutarate dehydrogenase, isocitrate lyase, phosphateacetyltransferase, acetate kinase, acetohydroxy acid synthase,acetolactate synthase, formate acetyltransferase, lactate dehydrogenase,glutamate decarboxylase, 1-pyrroline dehydrogenase and so forth.

L-Proline-producing ability can be imparted by, for example, making thebacterium have γ-glutamyl kinase desensitized in feedback inhibition byL-proline, and/or by destroying the L-proline degradation system. Themethod for making the bacterium have γ-glutamyl kinase desensitized infeedback inhibition by L-proline is exemplified by a method comprisingintroducing a DNA coding for γ-glutamyl kinase desensitized in feedbackinhibition by L-proline into cells (J. Bacteriol. 170, 5943 (1988)). Themethod for destroying the L-proline degradation system is exemplified bya method comprising introducing a mutation in a proline dehydrogenasegene so that no active proline dehydrogenase is expressed. Also, thebacterium in which the L-proline degradation system is destroyed can beobtained by obtaining a strain deficient in L-proline-assimilatingability and selecting a strain extracellularly produce L-proline fromthe obtained strains by using L-proline auxotrophy as an index.

L-Arginine-producing ability can be imparted by, for example, impartingresistance to α-methylmethionine, p-fluorophenylalanine, D-arginine,arginine hydroxamate, S-(2-aminoethyl)-cysteine, α-methylserine,β-2-thienylalanine or sulfaguanidine (Japanese Patent ApplicationLaid-Open No. 56-106598), or introducing an argA gene coding forN-acetylglutamate synthase (Japanese Patent Application Laid-Open No.57-5693).

<2> Method of the Present Invention

The method of the present invention comprises culturing the bacterium ofthe present invention in a medium containing L-isoleucine, to produceand accumulate L-glutamic acid, L-proline or L-arginine in a culture andcollecting L-glutamic acid, L-proline or L-arginine from the culture.

The medium may be an ordinary medium containing a carbon source, anitrogen source, inorganic ions and optionally other organic components,provided that it contains L-isoleucine. The amount of L-isoleucine isone sufficient to allow the bacterium of the present invention toproduce and accumulate L-glutamic acid, L-proline or L-arginine, and isusually 25 to 250 mg/l.

As the carbon source, it is possible to use sugars such as glucose,lactose, galactose, fructose, and starch hydrolysate; alcohols such asglycerol and sorbitol; or organic acids such as fumaric acid, citricacid and succinic acid.

As the nitrogen source, it is possible to use inorganic ammonium saltssuch as ammonium sulfate, ammonium chloride and ammonium phosphate;organic nitrogen such as soybean hydrolysate; ammonia gas; or aqueousammonia.

It is preferable to allow required substances such as vitamin B₁ oryeast extract to be contained in appropriate amounts as organic tracenutrients. Other than the above, potassium phosphate, magnesium sulfate,iron ion, manganese ion and the like are added in small amounts, ifnecessary.

Cultivation is preferably carried out under an aerobic condition for 16to 72 hours. The cultivation temperature is controlled at 25° C. to 45°C., and pH is controlled at 5 to 8 during cultivation. Inorganic ororganic, acidic or alkaline substances as well as ammonia gas or thelike can be used for pH adjustment.

The culture includes a medium and cells, and is preferably a medium.

Collection of L-glutamic acid, L-proline or L-arginine from the culturemay be usually carried out by combining an ion exchange resin method, aprecipitation method and other known methods.

EXAMPLES

The present invention will further specifically be explained withreference to the following examples hereafter.

Example 1 Production of L-glutamic Acid by ilvA Deficient Strain

A) Utilization of an Insertion of Transposon Tn5 (or any other) into theilvA Gene

Cells of the E. coli strain K12 of wild type (VKPM. B-7) were treatedwith a bacteriophage P1 which was grown on cells of L-isoleucineauxotrophic strain E. coli C600 ilvA::Tn5 having the insertion oftransposon Tn5 into the ilvA gene, and placed on LB agar plates,containing kanamycin (20 μg/ml), for selection of kanamycin resistanttransductants. As a result, a derivative of the wild type strain of E.coli K12 having insertion of transposon Tn5 into the ilvA gene wasobtained. This strain was named B7ILE, and has been deposited in theRussian National Collection of Industrial Microorganisms (VKPM) sinceJul. 18, 2000 and converted to a deposit under the Budapest Treaty onMay 18, 2001, and the accession number VKPM B-8013 is given.

B) Construction of an ilvA Deficient Derivative from the Wild TypeStrain E. coli K12, having a Mutation in the ilvA Gene

The strain VL334 (VKPM B-1641) is an L-isoleucine and L-threonineauxotrophic strain having mutations in thrC and ilvA genes (U.S. Pat.No. 4,278,765). A wild type allele of thrC gene was transferred by themethod of general transduction using a bacteriophage P1 grown on cellsof the wild type E. coli strain K12 (VKPM B-7). As a result, anL-isoleucine auxotrophic strain VL334thrC⁺ was obtained.

C) Production of L-glutamic Acid by the L-isoleucine Auxotrophic Strainin Test-tube Fermentation

The fermentation medium contained 60 g/l glucose, 25 g/l ammoniumsulfate, 2 g/l KH₂PO₄, 1 g/l MgSO₄, 0.1 mg/l thiamine, 50 mg/lL-isoleucine and 25 g/l chalk (pH 7.2). Glucose and chalk weresterilized separately. 2 ml of the medium was placed into test tubes,and inoculated with one loop of the tested microorganisms, and thecultivation was carried out at 37° C. for 2 days with shaking. Theresults are shown in Table 1.

TABLE 1 Accumulation of L-glutamic acid Strain Phenotype (g/l) K12 (VKPMB-7) Wild type <0.1 B7ILE (VKPM B-8013) IlvA::Tn5 2.0 VL334thrC⁺ IlvA44212.0

Example 2 Production of L-proline by an ilvA Deficient L-prolineProducer

The cells of wild type strain E. coli K12 (VKPM B-7) was treated with amutagen, N-methyl-N′-nitro-N-nitrosoguanidine (0.1 mg/ml), for 20 min at37° C., washed and plated on minimal agar medium M9 supplemented with1.25 mg/ml tryptone, 10 mg/ml L-proline and 0.05 mg/ml2,3,5-triphenyltetrazolium chloride. Most colonies arisen after 3 day ofincubation at 37° C. were colored red. A few colonies, which could notoxidize L-proline, were white. One of such colonies was used as a parentfor obtaining mutants resistant to proline analogs (3,4-dehydroxyprolineand azetidine-2-carboxylate) which were added into M9 agar medium inconcentration of 2 mg/ml each.

Some of mutants arisen could produce L-proline. The best L-prolineproducer 702 was treated with a P1 bacteriophage grown on cells of thestrain TG1 in which the gene ilvA was disrupted by the insertion ofchloramphenicol (Cm) resistance (Cm^(r)) gene. One of obtained Cmresisitant transductant, 702ilvA, which turned to be L-isoleucineauxotroph, was much more effective L-proline prouducer than theL-isoleucine prototrophic parent strain 702 (Table 2). The fermentationwas carried out as indicated in Example 1.

TABLE 2 Accumulation of Strain Phenotype L-proline (g/l) K12 (VKPM B-7)Wild type <0.1 702 (VKPM B-8011) Defective L-proline 0.5 degradation,resistance to proline analogs 702ilvA (VKPM Defective L-proline 8B-8012) degradation, resistance to proline analogs, L-isoleucineauxotroph, Cm^(r)

The strains 702 and 702ilvA have been deposited in the Russian NationalCollection of Industrial Microorganisms (VKPM) since Jul. 18, 2000 andconverted to a deposit under the Budapest Treaty on May 18, 2001 and theaccession numbers VKPM B-8011 And VKPM B-8012 are given, respectively.

Example 3 Production of L-arginine by an ilvA Deficient L-arginineProducer

The strain 237, an L-arginine-producing strain, which have been selectedas mutant resistant to a pyrimidine analog, 6-azauracil, has insertionof the transposon Tn5 into the ilvA gene and, therefore, it is anL-isoleucine auxotroph. The strain 237 has been deposited in the RussianNational Collection of Industrial Microorganisms (VKPM) since Apr. 10,2000 and converted to a deposit under the Budapest Treaty on May 18,2001, and the accession number VKPM B-7925 is given.

Cells of the strain 237 were treated with a P1 bacteriophage grown oncells of the wild type E. Coli K12 strain (VKPM B-7), and L-isoleucineprototrophic transformants were selected. The L-arginine production ofall L-isoleucine prototrophic transductants was drastically decreased(Table 3). The fermentation was carried out as indicated in Example 1.

TABLE 3 Accumulation of Strain Phenotype L-arginine (g/L) K12 (VKPM B-7)Wild type <0.1 237 (VKPM B-7925) Resistant to 4.5 6-azauracil,L-isoleucine auxotroph, ilvA::Tn5, Km^(r) 237ilvA⁺ Resistant to 0.4-0.66-azauracil, ilvA⁺

1. A method for producing L-glutamic acid, L-proline or L-arginine,which comprises culturing an Escherichia bacterium, which isL-isoleucine auxotrophic and has ability to produce L-glutamic acid,L-proline or L-arginine, in a medium containing L-isoleucine, to produceand accumulate L-glutamic acid, L-proline or L-arginine in a culture andcollecting L-glutamic acid, L-proline or L-arginine from the culture,wherein said collecting L-glutamic acid, L-proline or L-arginine fromthe culture is performed by an ion exchange resin method, precipitationmethod or combination thereof.
 2. The method according to claim 1,wherein the Escherichia bacterium is deficient in any of L-isoleucinebiosynthetic enzyme activities.
 3. The method according to claim 2,wherein the Escherichia bacterium is deficient in threonine deaminaseactivity.
 4. The method according to claim 1, wherein the Escherichiabacterium is Escherichia coli.